Leishmania are transmitted to human and other vertebrate hosts by sand flies. These parasites have a tremendous impact on human and animal health, particularly in developing countries. Resistance of the parasite to complement-mediated lysis (CML) is important for infection of vertebrates. CML is a microbe-induced host defense mechanism involving the cell free fraction of serum. In the sand fly, Leishmania develop from procyclic promastigotes that are not infectious to vertebrates into highly infectious metacyclic promastigotes. Resistance to CML is low in procyclic promastigotes, but is high in metacyclic promastigotes for most Leishmania species. The long term objective of this research is to understand the process by which Leishmania become infectious. The overall aim of this proposal is to identify and characterize genes that function in resistance to CML. Low passage metacyclic promastigotes of Leishmania chagasi are CML-resistant and have high levels of surface protein GP46, while high passage promastigotes, and low passage procyclic promastigotes are sensitive to CML and downregulate GP46 expression. High passage cells transfected with plasmids encoding GP46 were CML-resistant, indicating that high passage cells can be used to screen for specific Leishmania genes/coding sequences that confer resistance to CML. The high passage cells were used to screen Leishmania genomic DNA segments (32-35 kb fragments in cosmid vectors) for the ability to confer CML-resistance. A screen of five genomic equivalents resulted in selection of twelve cosmids. When re-transfected into CML-sensitive promastigotes, the selected cosmids conferred CML-resistance. The specific aims of this project are to determine and characterize the genes within the cosmid inserts that confer CML-resistance, and to characterize the mechanism by which the cosmids confer resistance to CML. Genes conferring CML resistance will be identified by sequence analysis and by screening subcloned inserts for functionality, and their biological relevance will be supported/tested by characterizing their gene expression profiles by using northern and western blot analyses. Characterization of the mechanism by which the cosmids (or genes) act will identify the point in the CML-cascade that is perturbed by the cosmids, and will study possible interactions between complement proteins and Leishmania proteins that confer CML-resistance.